The present invention relates to a method for detecting Alzheimer""s disease in vivo using magnetic resonance imaging.
Alzheimer""s disease is a widespread progressive dementia afflicting a signification proportion of the elderly population. While there has been significant research into the causes and treatment of Alzheimer""s disease, the primary pathology of the disorder remains unknown. The behavioral symptoms of Alzheimer""s disease are well known, and include loss of memory and cognitive function. The salient pathological symptom of Alzheimer""s disease at autopsy is the presence in certain brain areas of extracellular proteinaceous deposits or plaques called amyloid on the basis of their staining with various reagents.
To date, a definitive diagnosis of Alzheimer""s disease requires histopathological examination of brain tissue, usually at autopsy. The diagnosis is confirmed by the presence of amyloid plaques in sufficient numbers. Diagnostic clinical criteria have been developed for the disease, and when these are applied in research centers, diagnostic accuracy approaches 90%.
Unfortunately, there are no specific diagnostic tests for Alzheimer""s disease, and clinical diagnosis is most difficult in the earliest stages of the disease, when the few available treatments would be most helpful. There is a great need for reliable in vivo non-invasive diagnostic markers of disease and of disease progression. Of course, the most important would be non-invasive measurement of plaques and tangles within the brain. The need for such a methodology has become all the greater recently, with the development of several potential treatment strategies that enhance amyloid clearance. These are being tested in animals or are in Phase I clinical trials currently. For greatest efficacy, these methods will require the definitive identification of patients at early or pre-clinical stages of the disease and a method for monitoring amyloid burden.
In the absence of any method for detecting the histopathological hallmarks of the disease, short of cerebral biopsy, there have been a number of attempts to develop in vivo diagnostic techniques. Generally, these have involved imaging downstream events, such as metabolic changes with functional imaging, or cell loss with structural imaging. Structural imaging, computed tomography or magnetic resonance imaging, is now routinely used in clinical practice when a patient is suspected of having Alzheimer""s disease. Magnetic resonance imaging provides higher resolution and better tissue contrast than computed tomography, and may show characteristic hippocampal atrophy. Serially acquired magnetic resonance scans show accelerated cerebral tissue loss in Alzheimer""s disease relative to normal ageing. However, although these imaging techniques may help to distinguish the patient with a degenerative cause for the cognitive impairment from a normal control, they are much less specific at identifying the underlying molecular pathology.
Benveniste et al., in Proc. Natl. Acad. Sci. USA 96, 14079-14084 (1999) have applied magnetic resonance imaging, which has proven successful for detecting macroscopic changes such as atrophy, to the detection of microscopic molecular pathology. The potential targets for magnetic resonance microscopy are the plaques and tangles initially seen by Alois Alzheimer when he first diagnosed this disease. However, Benveniste et al. did not use a contrast agent, and to achieve the required resolution of 5.9xc3x9710xe2x88x925 xcexcm, scanning times of about 20 hours were needed, something that is not feasible for in vivo studies. Furthermore, a recent attempt to reproduce these findings using even better MRI resolution on brain samples from AD patients, concluded that amyloid could not be imaged directly (Dhenain et al., NMR Biomed. 15: 197-203, 2002).
The magnetic resonance microscopy techniques Benveniste et al. used involved a 7-T machine with coils of 1 cm to image formalin-fixed tissue sections 1 cm in diameter. To visualize the plaques, the contrast-to-noise ratio must be maximized. Two acquisition techniques were used for this: diffusion weighted imaging and T2 imaging. Diffusion-weighted imaging, which measures how well water can undertake Brownian motion, did not show the plaques, suggesting that the plaques did not provide a substantial obstruction to the movement of water molecules. T2 imaging, which measures local perturbations to the applied magnetic field, did show some plaques, but, as noted above, required a resolution of 5.9xc3x9710xe2x88x925 mm3 and required scanning times of about 20 hours. Such long scanning times would not be feasible in vivo. In addition, this imaging was performed on excised tissue, not intact brains.
The neurofibrillary tangles are intraneuronal aggregates of abnormally phosporylated tau, the microtubule binding protein. The neuritic plaque consists of a central core of amyloid xcex2-protein (Axcex2) fibrils surrounded by dystrophic neuritis (abnormal axons and dendrites). These plaques are extracellular, roughly spherical in shape, and range from about 5 xcexcm to about 200 xcexcm in diameter. The histopathological changes and neuronal loss are particularly severe in the hippocampus and medial temporal lobe, where the disease is believed to start, and in association areas of the neocortex. This regional selectivity determines the characteristic clinical feature of early memory loss. Whether tangles or plaques are more relevant to the pathogenesis of Alzheimer""s disease is a matter of some debate.
Senile plaques are a more promising target than neurofibrillary tangles for visualizing with magnetic resonance imaging because they are extracellular and larger. To visualize even the largest plaques pushes MRI to its very limit. Conventional clinical magnetic resonance imaging employs magnets with field strengths ranging from about 0.5 T to about 1.5 T. With an acquisition time of less then seven minutes, a 1.5 T machine can image the entire brain with good grey matter/white matter contrast and a voxel size of 1 mm3. In this way, small cerebral structures such as the hippocampus can be fairly reliably measured in vivo, and subfields of the hippocampus can be resolved. Plaques, however, are an order of magnitude smaller than the best depiction of brain structure achievable in vivo.
It is an object of the present invention to overcome the aforesaid deficiencies in the prior art.
It is another object of the present invention to provide an in vivo method for diagnosing Alzheimer""s disease.
It is a further object of the present invention to detect amyloid plaques in vivo using magnetic resonance micro-imaging.
It is yet another object of the present invention to use labeled peptides for imaging preamyloid deposits and Congo red positive amyloid plaques.
It is another object of the present invention to provide a method for an early definite diagnosis of Alzheimer""s disease.
It is still a further object of the present invention to provide a method for monitoring amyloid clearing in vivo.
The present invention provides a method for detecting the amyloid plaques characteristic of Alzheimer""s disease using magnetic resonance micro-imaging (xcexcMRI). The method uses Axcex2-1-40 peptides, derivatives or mutants thereof or Axcex2 homologous peptides which are labeled with a label that enhances the image in MRI. Particularly useful labels are gadolinium, manganese or monocrystalline iron oxide nanoparticles. One of these ligands is injected systemically with mannitol or another compound which acts as a carrier and transiently opens the blood-brain barrier so that the majority of both early pre-Axcex2 deposits and Congo red positive amyloid plaques can be visualized.
An alternative strategy is to couple the labeled Axcex2 peptide to a carrier molecule that more readily crosses the blood-brain barrier (BBB). This can be accomplished in three different ways:
1. The labeled-Axcex2 peptide can have its lipid solubility increased by coupling the peptide to a cholesteryl group at the C- or N-terminus of the peptide (Bodor and Simpkins 1983; Science, 221: 65-67). Alternatively, the labeled-Axcex2 peptide can be coupled to putrescine, which increases the lipid solubility and BBB passage (T. M. Wegenack, G. L. Curran, and J. F. Poduslo, Nat. Biotech. 18:868-872, 2000).
2. Chimeric peptidization can be used, in which the labeled-Axcex2 peptide is coupled to a compound that has increased passage across the BBB, such as albumin (A. K. Kumagai, J. B. Eisenberg, and J. F. Poduslo, J. Biol. Chem. 262: 15214-15219, 1987).
3. The labeled-Axcex2 peptide can be coupled to an antibody that targets a receptor on the BBB, such as the transferrin receptor. The antibody-labeled-Axcex2 would then be carried across the BBB by transcytosis (W. M. Partridge, J. Buciak, and T. Yokishawa, J. Pharmacol. 269:66-70, 1991).
The method of the present invention makes it possible to diagnose Alzheimer""s disease at an early stage so that treatment can be started as soon as possible to mitigate the damage. Additionally, the present invention permits monitoring of amyloid clearing in vivo.